How to cite: Vineeth, M. R., Gupta, I. D., Verma, A., & Kumari, S. (2019). Screening of Sahiwal Bulls for Fertility-Related PROP1 Gene Isoforms. International Journal of Livestock Research, 9(10), 24-29. doi: 10.5455/ijlr.20190613041018

Introduction

The Prophet of Pit1 (PROP1) gene, a member of homeobox gene family and is located on chromosome 7 at location 41205895 to 41209913 bases, conferring a total length of 4019 bp. PROP1 comprises of 4 exons and 3 introns and encodes a paired class homeodomain transcription factor with a polypeptide length of 226 amino acids representing the prophet of Pit-1 transcription factor (also known as POU1F1 transcription factor) as it lies upstream of POU1F1 in pituitary development (Davis et al., 2010; Sornson et al., 1996). Thus, POU1F1 gene is a direct downstream target for the regulation of the PROP1 gene. Previous studies have found that PROP1 mutations affects the expression level of the POU1F1 gene, and thus the GH, PRL, and TSH-β expression levels (Carvalho et al., 2006; Davis et al., 2010). There are reports of associations of the POU1F1 pathway genes like POU1F1, GH, PRL, GHR, PRLR, STAT5A, OPN and UTMP with early embryonic survival, fertilization rate (Khatib et al., 2009) and male fertility (Khatib et al., 2010).

PROP1 gene takes part in the Wnt/b-catenin Signaling pathway (Olson et al., 2006). It includes nuclear recruitment of b-catenin and activation of Wnt-dependent transcription factors leading to development and differentiation of the diverse reproductive tissues and affects implantation, decidualisation and placental differentiation (Sonderegger et al., 2010). WNT signaling and b-catenin in the testis has been associated with proliferation and selfrenewal of spermatogonia (Golestaneh et al., 2009) and also in male infertility (Boyer et al., 2008). In addition, the bovine PROP1 gene is located at a QTL on chromosome 7 which affects ovulation rate in cattle (Kappes et al., 2000; Kirkpatrick et al., 2000).

Recent studies found a missense single nucleotide polymorphism (rs136195618 A>G) that replaces a histidine amino acid with an arginine (H173R) in exon 3 of bovine PROP1 gene. The bovine isoform PROP1-173H, representing the “A” allele strongly activates transcription of the reporter gene and the PROP1-173R isoform was reported to be associated with a 5-fold reduction in binding capacity as well as a reduced activation capacity, when compared to the bPROP1-173H carboxyl domain (Showalter et al., 2002). The PROP1-173R isoform was found to be associated with a decrease in sire conception rate and an increase in productive life and protein yield in Holstein bulls (Lan et al., 2013). The PROP1-173R isoforms were found to have positive effect on growth traits in cattle (Pan et al., 2013).

Till date, it remains unknown whether there exist PROP1 gene isoforms in indigenous cattle breeds of India. Therefore, the present study was carried out with the objective to characterise the exon 3 of PROP1 gene and to screen H173R (rs136195618 A>G) polymorphism so as to identify the PROP1 gene isoform related to bull fertility.

Materials and Methods

Sample Collection and DNA Isolation

Frozen semen straws of sixty-two Sahiwal bulls were collected from Artificial Breeding Research Centre (ABRC) of National Dairy Research Institute, Karnal and were utilized for isolation of genomic DNA. The DNA isolation was performed using a protocol that involves two steps: Lysis and extraction. Lysis of spermatozoa was done as per Hossain et al. (1997), while the extraction of DNA was done by standard Phenol Chloroform extraction procedure (Sambrook and Russell, 2001). The concentrations of DNA were measured using Biospec-nano-spectrophotometer (Schimadzu Cooperation, Japan).

Primer Designing

In silico primer designing for exon3 of bovine PROP1 gene was carried out using Primer3 software (http://www.primer3.ut.ee) (Untergrasser et al., 2012) using Bos taurus sequence (GenBank Ref Seq: AC_000164.1) as reference.

PCR Amplification

PCR amplification was carried out in a total volume of 25 ml with 100 ng DNA template, 1x PCR buffer, 1.5 mM MgCl2, 200 mM of each dNTPs, 20 pmol of each primer and 1 unit of Taq DNA polymerase. The PCR programme was initial denaturation at 95°C for 5 min. The cycling protocol was denaturation at 95°C for 30 sec, then annealing temperature of 59°C for 30 sec then extension at 72°C for 30 sec followed by final extension at 72°C for 5 min. The number of cycles was 35. Quality and sizes of PCR products were checked on 2% agarose gel electrophoresis (Fig. 1).

Fig. 1: Resolution of PCR product of exon3; L=100bp ladder

Sequencing and SNP Screening

PCR products were stored at -20ºC and sent to first BASE Sequencing INT, Singapore, for direct sequencing from both 5′ and 3′ ends. The raw sequences obtained after sequencing were edited using BioEdit software. Sequencing results were screened for (rs136195618 A>G) polymorphism and other reported/novel SNPs by ClustalW alignment analysis with Bos taurus reference sequence: AC_000164.1 (NCBI), and confirmed by visual inspection of chromatograms.

BLAST Analysis

Analysis of the sequence results was done to find homology with other species using BLASTN (http:/ /blast.ncbi.nlm.nih.gov/). Phylogram was also constructed using online tool phylogeny.fr [(http://www.phylogeny.fr/) (Dereeper et al., 2008)].

Results and Discussion

The third exon of PROP1 gene was characterised in Sahiwal by DNA sequencing and the nucleotide sequence was submitted to GenBank and accession number ‘KT198685.1’ was obtained. BLAST analysis of the Sahiwal exon 3 nucleotide sequence showed 100% sequence identity with Karan Fries and Bos taurus. Phylogram of exon 3 of PROP1 (Fig. 2) shows that the Sahiwal is closely related to Bos taurus and Karan Fries in evolutionary tree whereas it is distantly placed from rodents.

Fig. 2: Phylogram of Exon 3 of PROP1gene

Fig. 3: Chromatogram showing A allele at rs136195618 locus (PROP1-173H isoform) in Sahiwal bulls

ClustalW multiple alignment analysis with Bos taurus (NCBI Ref Seq: AC_000164.1) did not found any nucleotide variation in the targeted region. Screening of SNP locus (rs136195618) showed that the reference allele A was present in all the animals included in the present study (Fig. 3). Thus, the studied bulls possessed the bovine PROP1-173H isoform. The SNP (rs136195618 A>G) was observed in different cattle breeds like German Holstein Friesian, US Holstein Friesian, German Flekvieh and indigenous cattle breeds of China. (Lan et al., 2013; Pan et al., 2013; Pausch et al., 2015). Despite the fact that Karan Fries is having Holstein Friesian blood the H173R polymorphism was not detected in the Karan Fries bulls (Vineeth et al., 2017). No reports are available about the H173R polymorphism status in other indigenous cattle breeds of India.

Conclusion

The third exon of bovine PROP1 gene has been characterized for the first time in Sahiwal bulls for which NCBI GenBank accession number KT198685.1 was obtained. The screening for fertility related PROP1 isoforms revealed that the indigenous bull population under the present study was having PROP1-173H isoform, the one which is favorable for bull fertility. The results obtained in the present study emphasize the importance of prior screening of candidate functional SNPs in population before using them in selection programmes.

Acknowledgements

The authors gratefully acknowledge Director, ICAR-NDRI, Karnal and Head, Animal Genetics and Breeding Division for providing necessary research facilities. In charge, ABRC is acknowledged for providing the semen straw samples for the study.

References

1. Boyer, A., Hermo, L., Paquet, M., Robaire, B., & Boerboom, D. (2008). Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells. Biology of Reproduction, 79(3), 475-485.
2. Davis, S. W., Castinetti, F., Carvalho, L. R., Ellsworth, B. S., Potok, M. A., Lyons, R. H., & Hayashizaki, Y. (2010). Molecular mechanisms of pituitary organogenesis: in search of novel regulatory genes. Molecular and Cellular Endocrinology, 323(1), 4-19.
3. DeJarnette, J. M., Marshall, C. E., Lenz, R. W., Monke, D. R., Ayars, W. H., & Sattler, C. G. (2004). Sustaining the fertility of artificially inseminated dairy cattle: the role of the artificial insemination industry. Journal of Dairy Science, 87, E93-E104.
4. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., & Claverie, J. M. (2008). Phylogeny. fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research, 36(suppl_2), W465-W469.
5. Golestaneh, N., Beauchamp, E., Fallen, S., Kokkinaki, M., Uren, A., & Dym, M. (2009). Wnt signaling promotes proliferation and stemness regulation of spermatogonial stem/progenitor cells. Reproduction, 138(1), 151.
6. Hossain, A. M., Rizk, B., Behzadian, A., & Thorneycroft, I. H. (1997). Modified guanidinium thiocyanate method for human sperm DNA isolation. Molecular Human Reproduction, 3(11), 953-956.
7. Kappes, S. M., Bennett, G. L., Keele, J. W., Echternkamp, S. E., Gregory, K. E., & Thallman, R. M. (2000). Initial results of genomic scans for ovulation rate in a cattle population selected for increased twinning rate. Journal of Animal Science, 78(12), 3053-3059.
8. Khatib, H., Huang, W., Wang, X., Tran, A. H., Bindrim, A. B., Schutzkus, V., … & Yandell, B. S. (2009). Single gene and gene interaction effects on fertilization and embryonic survival rates in cattle. Journal of Dairy Science, 92(5), 2238-2247.
9. Khatib, H., Monson, R. L., Huang, W., Khatib, R., Schutzkus, V., Khateeb, H., & Parrish, J. J. (2010). Validation of in vitro fertility genes in a Holstein bull population. Journal of Dairy Science, 93(5), 2244-2249.
10. Kirkpatrick, B. W., Byla, B. M., & Gregory, K. E. (2000). Mapping quantitative trait loci for bovine ovulation rate. Mammalian Genome, 11(2), 136-139.
11. Lan, X. Y., Peñagaricano, F., DeJung, L., Weigel, K. A., & Khatib, H. (2013). A missense mutation in the PROP1 (prophet of Pit 1) gene affects male fertility and milk production traits in the US Holstein population. Journal of Dairy Science, 96(2), 1255-1257.
12. Olson, L. E., Tollkuhn, J., Scafoglio, C., Krones, A., Zhang, J., Ohgi, K. A., … & Rose, D. (2006). Homeodomain-mediated β-catenin-dependent switching events dictate cell-lineage determination. Cell, 125(3), 593-605.
13. Pan, C., Wu, C., Jia, W., Xu, Y., Lei, C., Hu, S., … & Chen, H. (2013). A critical functional missense mutation (H173R) in the bovine PROP1 gene significantly affects growth traits in cattle. Gene, 531(2), 398-402.
14. Pausch, H., Wurmser, C., Reinhardt, F., Emmerling, R., & Fries, R. (2015). Validation of 4 candidate causative trait variants in 2 cattle breeds using targeted sequence imputation. Journal of Dairy Science, 98(6), 4162-4167.
15. Sambrook, J., & Russell, D. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
16. Showalter, A. D., Smith, T. P., Bennett, G. L., Sloop, K. W., Whitsett, J. A., & Rhodes, S. J. (2002). Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein. Gene, 291(1-2), 211-221.
17. Sonderegger, S., Pollheimer, J., & Knöfler, M. (2010). Wnt signalling in implantation, decidualisation and placental differentiation–review. Placenta, 31(10), 839-847.
18. Sornson, M. W., Wu, W., Dasen, J. S., Flynn, S. E., Norman, D. J., O’connell, S. M. & Zuo, L. (1996). Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature, 384(6607), 327.
19. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen, S. G. (2012). Primer3—new capabilities and interfaces. Nucleic Acids Research, 40(15), e115-e115.
20. Vineeth, M. R., Gupta, I. D., Verma, A., Magotra, A., Kumar, R., Verma, N., & Kumari, S. (2017). Characterization of Exon 3 of PROP1 gene and screening of H173R polymorphism in Karan Fries bulls. Indian Journal of Animal Research, 51(2), 275-279.